CN104492396A - Targeted liposome based on protein molecular imprinting and preparation method thereof - Google Patents
Targeted liposome based on protein molecular imprinting and preparation method thereof Download PDFInfo
- Publication number
- CN104492396A CN104492396A CN201410755690.1A CN201410755690A CN104492396A CN 104492396 A CN104492396 A CN 104492396A CN 201410755690 A CN201410755690 A CN 201410755690A CN 104492396 A CN104492396 A CN 104492396A
- Authority
- CN
- China
- Prior art keywords
- preparation
- liposome
- ultrasonic
- spa
- protein molecular
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Medicinal Preparation (AREA)
Abstract
The invention discloses a targeted liposome based on protein molecular imprinting and a preparation method thereof. The preparation method of the targeted liposome based on the protein molecular imprinting comprises the following concrete steps: dissolving an acrylamide compound and N-N methylene bisacrylamide into PBS (phosphate buffer solution) or water, and carrying out ultrasonic treatment, thus mixed solution A is obtained; then modifying a silicon dioxide liposome by adopting an olefinic bond, dispersing protein molecules in olefinic bond modified silicon dioxide lipidosome solution, stirring, and carrying out vacuum degassing, thus mixed solution B is obtained; dropwise adding the mixed solution A into the mixed solution B in presence of nitrogen, blowing nitrogen, stirring, carrying out pre-polymerization, then dropwise adding ammonium persulphate and tetramethylethylenediamine in presence of nitrogen for initiating polymerization, continuously stirring, and dialyzing, thus the targeted liposome based on the protein molecular imprinting is obtained. The prepared targeted liposome based on the protein molecular imprinting takes a silicon dioxide modified liposome as a core and an acrylamide compound and N,N'-methylene bisacrylamide as a shell, target protein of a focus part can be initiatively recognized, and the targeted liposome based on the protein molecular imprinting can be applied to mediated active targeting drug delivery.
Description
Technical field
The invention belongs to chemical field, be specifically related to the preparation method of the target liposomes based on protein molecular trace, also relate to product obtained by this method.
Background technology
Molecular imprinting (molecular imprinting technique, MIT), also known as molecular imprinting, molecular imprinting technique or molecular templating techniques, it is a kind of emerging molecular recognition technology, its objective is that preparation has the polymer of specific selectivity recognition capability to template molecule, i.e. molecularly imprinted polymer (MIPs).Molecular imprinting is developed so far, and compared to reaching its maturity of Small molecular engram technology, protein and other engram technology also relatively lags behind.The specific binding in conjunction with similar " antigen-antibody " of molecular engram material and template protein, imprinted material can keep stable in high temperature, high pressure, extreme pH condition and organic reagent, be convenient to store, so research and development can identification of protein thus the molecular engram " man-made antibody " of simulating nature process has very large potential using value in physiological conditions.
Compared with traditional blotting techniques, surface molecule print is in material surface because of the recognition site preparing resulting polymers, effectively can improve the template molecule that conventional method causes excessively to embed, the problem such as not easily to remove, this is for macromolecule blot important in inhibitings such as protein.For making gained imprinted polymer particle, homogenization (microballoon, nanoparticle etc.), researchers often adopt nucleocapsid structure material to realize surface imprinted target, that is: with previously prepared good microballoon or nanoparticle as support matrix (" core "), the imprinted polymer covering surfaces that it is formed is " shell ".The trace strategy of this nucleocapsid structure in turn introduces the new material of a class outside the function monomer needed for usual trace, i.e. support matrix material.Earth silicon material has the features such as surface is easy to modify, stability is strong, chemical/biological inertia, particle diameter and dispersiveness are controlled and it is applied to biomedicine field already, therefore, silica is the ideal support matrix material of a class, relies on silicon dioxide granule to carry out and is surface imprintedly expected to realize to effective identification of template protein and has good potential using value.
Realize the important channel that active targeting passs medicine by mediating in pharmaceutical carrier finishing for the part of specified disease tissue target molecule (mostly being protein) and improve the identification of drug-loading system to target area.Because biological species molecule (as polypeptide, antibody, aptamer etc.) often faces the problem of body internal stability, and surface molecule print has the potentiality of " artificial antibody "/" artificial ligand " and imprinted sites stability is strong, therefore, carry out for disease association target protein surface imprinted likely for active targeting passs the means that medicine provides new.
Summary of the invention
In view of this, an object of the present invention is the preparation method of the target liposomes provided based on protein molecular trace, and this preparation method is simple, quick, cost is low; Two of object of the present invention is to provide the target liposomes based on protein molecular trace obtained by said method.
For achieving the above object, the invention provides following technical scheme:
Based on the preparation method of the target liposomes of protein molecular trace, comprise the steps: to get acrylamides and N-N methylene-bisacrylamide is in the PBS solution or water that 1 ~ 5:1 is dissolved to pH6.8 ~ 7.4, concentration is 0.02 ~ 0.1M in mass ratio, ultrasonic, obtain mixed liquor A; Olefinic recycle key modifies silica liposome, and be then scattered in by protein molecular in the silica liposome solutions of ethylene linkage modification and stir, vacuum outgas obtains mixed liquid B; Under nitrogen protection, mixed liquor A is added dropwise in mixed liquid B for 1:1 ~ 1:10 by volume, nitrogen blowing, stirs prepolymerization; then ammonium persulfate and tetramethylethylenediamine initiated polymerization is dripped under nitrogen protection; continue to stir, dialysis, must based on the target liposomes of protein molecular trace.
In the present invention, acrylamides is preferably NIPA or acrylamide, and acrylamides and N-N methylene-bisacrylamide mass ratio are preferably 2:1; PBS solution is preferably pH 7.4, concentration is the PBS solution of 0.02M.
Preferably, the preparation method of described silica liposome is: first adopt film dispersion ultrasonic method to prepare liposome, then in liposome, add the ethyl orthosilicate through ethanolic solution dispersion, pH to 8 ~ 10.4 are regulated with sodium hydroxide solution under stirring, stirring is spent the night, dialysis, obtains silica liposome.
Preferred, the addition of described ethyl orthosilicate presses phosphatide: ethyl orthosilicate: H
2the mol ratio of O is 1:8:344.
Preferred, the volume fraction of described ethanolic solution is 5% ~ 25%.
Preferably, the method that described film dispersion ultrasonic method prepares liposome be phosphatide and cholesterol are dissolved in volume ratio be the chloroform of 1:1 with the mixed solution of methyl alcohol, decompression rotary evaporation removes organic solvent, form transparent lipid membrane, add water hydratable, ultrasonic, obtain liposome.
Preferred, the mass ratio of described phosphatide and cholesterol is 7:3, and the liposome formed under this ratio is more conducive to follow-up modification.
In the present invention, phosphatide is preferably soybean lecithin or egg yolk lecithin.
Preferably, described ultrasonic condition is under 800W condition ultrasonic for 50 times, and each ultrasonic 10s, continues ultrasonic after the 10s of interval.
Preferably, the condition of described aquation is 37 DEG C of Water Underizations 1 hour.
Preferred, described protein molecular is staphylococcus aureus protein A or matrix metalloprotease MMPs.
2, the target liposomes based on protein molecular trace obtained by described preparation method.
Beneficial effect of the present invention is: the preparation method that the invention discloses the target liposomes based on protein molecular trace, preparation method is simple, cost is low, fast, obtained protein molecular imprinted polymer is using liposome as central support, utilize the original medicine carrying of liposome and pass medicine advantage, and particle diameter is easy to control; Owing to having modified layer of silicon dioxide film in the periphery of liposome, the stability of medicine can be improved, reduce the impact of the unfavorable factors such as acidity, temperature and enzyme and there is medicament slow release ability, this external silicon materials surface is by introducing ethylene linkage, albumen and monomer material crosslinking copolymerization can be made in shell surface, make the monomer material amount of input low, can also avoid being polymerized the gelation that rear dispersion easily occurs, and make the trace particle more easily collecting of not reuniting, surface imprinted better to realize, liposome after trace is used for medicine carrying, the targeting of liposome can be improved.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:
Fig. 1 is the silicon dioxide modified liposome (S-LIP) of infrared spectrum measurement, ethylene linkage modify after silica liposome (MS-LIP) and (a is SPA-MIPs the architectural feature peak of target liposomes (SPA-MIPs) of staphylococcus aureus trace, b is MS-LIP, c is S-LIP).
Fig. 2 is target liposomes (SPA-MIPs) and non-imprinted polymer (NIPs) the Dynamic Adsorption experimental result picture of staphylococcus aureus trace.
Fig. 3 is target liposomes (SPA-MIPs) and non-imprinted polymer (NIPs) the adsorption isotherm experiment result figure of staphylococcus aureus trace.
Fig. 4 is the non-specific adsorption experimental result picture of the target liposomes (SPA-MIPs) of staphylococcus aureus trace.
Fig. 5 is that staphylococcus aureus is carried the SPA imprinted polymer (FAM-SPA-MIPs) of 6-Aminofluorescein to bag, wraps the picked-up experimental result picture of year non-imprinted polymer of 6-Aminofluorescein (FAM-NIPs) and 6-Aminofluorescein (6-FAM).
To be staphylococcus aureus carry the SPA imprinted polymer (FAM-SPA-MIPs) of 6-Aminofluorescein to bag to Fig. 6, bag carries the non-imprinted polymer of 6-Aminofluorescein (FAM-NIPs) and 6-Aminofluorescein (6-FAM) to the bacteriostatic experiment result figure of staphylococcus aureus.
Fig. 7 is the target liposomes (MMP-2-MIPs) of matrix metalloproteinase trace and the Dynamic Adsorption experimental result picture of non-imprinted polymer (NIPs).
Fig. 8 is the target liposomes (MMP-2-MIPs) of matrix metalloproteinase trace and the adsorption isotherm experiment result figure of non-imprinted polymer (NIPs).
Fig. 9 is the target liposomes (MMP-2-MIPs) of matrix metalloproteinase trace and the specific adsorption experimental result picture of non-imprinted polymer (NIPs).
Figure 10 is that U937 cell carries the matrix metalloproteinase MMP-2 imprinted polymer (DOX-MMP-2-MIPs) of adriamycin and the picked-up experimental fluorescence microscopy images figure of the non-imprinted polymer (DOX-NIPs) of bag year adriamycin to bag.
Detailed description of the invention
Below in conjunction with accompanying drawing, the preferred embodiment of the present invention is described in detail.The experimental technique of unreceipted actual conditions in embodiment, the usually conveniently conditioned disjunction condition of advising according to manufacturer.
The preparation of the target liposomes of embodiment 1, staphylococcus aureus protein A (SPA) trace and evaluation thereof
The preparation method of the target liposomes of SPA trace, comprises the steps:
(1) film dispersion ultrasonic method is adopted to prepare liposome, concrete grammar is: take soybean lecithin 10.5mg, cholesterol 4.5mg, be that the chloroform of 1:1 and methyl alcohol mixed solution 1mL dissolve by volume ratio, then the rotary evaporation that reduces pressure under 37 DEG C of conditions removing organic solvent, form the uniform transparent lipid membrane of one deck, add pure water 1mL in 37 DEG C of aquation 1h, utilize ultrasonic cell disrupte instrument at 800W ultrasonic for 50 times, after each ultrasonic 10s, interval 10s continues ultrasonic, obtains liposome (LIP);
(2) adopt sol method to prepare the liposome of finishing silica, concrete grammar is: by phosphatide: ethyl orthosilicate (TEOS): H
2o mol ratio be 1:8:344 (mol/mol) slowly add in liposome through volume fraction be 10% ethanol dispersion TEOS, then pH to 8 ~ 10.4 are regulated with the sodium hydroxide solution of 0.1mol/L under agitation, room temperature (18-25 DEG C) stirs spends the night, the silica precursor do not deposited in removing system that mixture is dialysed, obtain the liposome of finishing silica, called after silica liposome (S-LIP);
(3) ethylene linkage modifies silica liposome, concrete grammar is: be slowly added drop-wise to by 3-sulfydryl-1-propane sulfonic acid sodium (MPS) of 50 μ L after disperseing half an hour in the ethanol of 250 μ L, then get 10 μ L dispersant liquid drops to be added in the liposome of 2ml silica room temperature (18-25 DEG C) and to stir and spend the night, obtain the silica liposome (MS-LIP) that ethylene linkage is modified;
(4) preparation of the target liposomes of SPA trace, concrete grammar is: getting acrylamide (AAM) 4mg and N-N methylene-bisacrylamide (MAA) 2mg, to be dissolved into 20ml pH be 7.4, concentration is in the PBS of 0.02M, ultrasonic dissolution, obtain mixed solution A, then 2mg SPA is dispersed in 1mL ethylene linkage modify silica liposome solutions in and be positioned over stirring at room temperature 0.5h, again mixture is carried out vacuum outgas 10min, obtain mixed solution B, getting 200 μ L mixed solution A is under nitrogen protection added dropwise in mixed solution B, and nitrogen blowing 10min, room temperature (18-25 DEG C) stirs lower prepolymerization 1h, 7 μ L ammonium persulfate (APS are dripped under nitrogen protection, 10%, W/W) and 4.5 μ L tetramethylethylenediamine (TEMED, 5%, W/V) initiated polymerization in mixture, and then be positioned over room temperature (18-25 DEG C) stirring 24h, with pure water dialysis one week, removing template molecule and unreacted reagent, freeze-drying is to constant weight and S. aureus L-forms surface protein SPA imprinted polymer (referred to as SPA-MIPs).
In the present embodiment, acrylamide directly can replace with NIPA, and the mass ratio of NIPA and N-N methylene-bisacrylamide is at 1 ~ 5:1, and the total concentration of NIPA and N-N methylene-bisacrylamide in mixed solution A is made to be 1 ~ 5mg/ml; During dispersion TEOS, the volume fraction of ethanol is 5% ~ 25%; PBS solution is pH6.8 ~ 7.4, concentration is all can realize goal of the invention within the scope of 0.02 ~ 0.1M.
The synthesis of non-imprinted polymer (NIPs) is not except adding template protein molecule, and other preparation methods are the same.
Preliminary characterization is carried out to the targeting preparation constructed by the present invention: take S-LIP and SPA-MIPs that prepare at 25 DEG C, adopted Malvern laser particle analyzer to measure its particle size and distribution situation, and replication three times.Result shows, and the average grain diameter of S-LIP is 147.9 ± 40.5nm, PI value is 0.214 ± 0.015, its dispersiveness is relatively good, and after trace polymerization, the average grain diameter of SPA-MIPs is 200.5 ± 60.5nm, PI value is 0.22 ± 0.018, show that SPA-MIPs preparation particle diameter is less, be evenly distributed.In addition, adopt the architectural feature peak of infrared spectrum measurement S-LIP, MS-LIP, SPA-MIPs, be specially: first by S-LIP, MS-LIP, SPA-MIPs respectively through freeze drying process, then KBr is mixed for 100:1 is placed in agate mortar in mass ratio with S-LIP, MS-LIP, SPA-MIPs respectively, finally roll, adopt Fourier infrared spectrograph to measure after compressing tablet, measurement result as shown in Figure 1.Result shows, and S-LIP is (c) at 1087cm in Fig. 1
-1there is the characteristic absorption peak of Si-O-Si key in place; MS-LIP is (b) at 1712cm in Fig. 1
-1the appearance at place is Si-C=C characteristic absorption peak, and the surface that shows silica is grafting ethylene linkage successfully.SPA-MIPs (in Fig. 1 a) after trace polymerization, 1712cm
-1the absworption peak at place disappears, and shows that trace type liposome SPA-MIPs is successfully prepared.
The adsorption experiment of the target liposomes of SPA trace: for investigating the SPA-MIPs for preparing to the specific adsorption ability of SPA, adopt following methods to verify it.
Dynamic Adsorption is tested: first carry out FITC fluorescence labeling to SPA, specifically get 4mg SPA and be dissolved in 0.2M Na
2pO
4in solution, separately get 0.16mg FITC and be dissolved in 0.2M Na
2pO
4in solution, the mol ratio making FITC and SPA is 1.2:1, under magnetic agitation, is added dropwise in the solution containing FITC by the solution containing SPA, lucifuge reaction 1.5h; After reaction terminates, under 8000r/min condition, centrifugal 10min is to remove precipitation SPA, gets supernatant Sephadex G-50 gel column and carries out separation and purification, freeze-drying, obtain FITC-SPA.Then by 5mg SPA-MIPs and NIPs respectively with the 0.2mg/ml FITC-SPA solution of 5ml in 37 DEG C of oscillation incubations, 1mL is taken out respectively at 0.25h, 0.5h, 1h, 2h and 4h, with the centrifugal 10min of 4500r/min, measure the fluorescence intensity of supernatant, result as shown in Figure 2.Result shows, and the absorption of SPA-MIPs and NIPs to FITC-SPA increases along with the increase of time, reaches dynamic equilibrium at 2h.Further, during Dynamic Adsorption balance, SPA-MIPs is about 5 times of NIPs to the adsorbance of FITC-SPA, illustrates that the molecular engram target polymer carrier that the present invention builds has well heavy adsorption capacity to albumen.
Adsorption isotherm experiment: accurately take 1mg SPA-MIPs and NIPs and be placed in centrifuge tube respectively, respectively with FITC-SPA solution oscillation incubation 2h under 37 DEG C of conditions of 0.05mg/ml, 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.8mg/ml of 1ml, with the centrifugal 10min of 4500r/min after hatching, Aspirate supernatant measures fluorescence intensity, and result as shown in Figure 3.Result shows, polymer is directly proportional to FITC-SPA concentration to the ratio of adsorption of template molecule, and when the concentration of FITC-SPA reaches 0.4mg/ml, ratio of adsorption reaches balance; Compared with NIPs, SPA-MIPs has higher adsorption capacity and selective preferably to FITC-SPA.
Non-specific adsorption is tested: choose matrix metalloproteinase MMP-2, lysozyme (LYZ), bovine serum albumin(BSA) (BSA), Streptavidin (SA) is competition albumen, carry out FITC fluorescence labeling according to the method described above, obtain FITC-MMP-2 respectively, FITC-LYZ, FITC-BSA, FITC-SA, then take 1mg SPA-MIPs and NIPs and be placed in centrifuge tube respectively, add the FITC-MMP-2 of the 0.2mg/ml of 1ml, FITC-LYZ, FITC-BSA, after FITC-SA and FITC-SPA solution to be placed on oscillator vibration absorption 2h in 37 DEG C, measure the fluorescence intensity in supernatant respectively, result as shown in Figure 4.Result shows, and SPA-MIPs only has higher absorption property to SPA, and relatively low to the absorption property of other four kinds competition albumen, suitable with the absorption property of NIPs to SPA.This illustrates that the imprinted polymer SPA-MIPs constructed by the present invention has higher specific selectivity to template molecule SPA.
On investigation basis on the target liposomes molecular level of SPA trace, also investigate in its bacteria levels.SPA albumen due to trace is a kind of protein be separated from aureus cell wall, is the main component of cell wall antigen.Therefore, first bag is carried out to the molecular engram targeted system constructed by the present invention and carry 6-Aminofluorescein (6-FAM), measure by fluidic cell the targeting that the picked-up of bacterium to different several preparations evaluates them.Bag carries the imprinted polymer (FAM-SPA-MIPs) of 6-Aminofluorescein and the target liposomes (SPA-MIPs) of the method for non-imprinted polymer (FAM-NIPs) with reference to SPA trace and the preparation method of non-imprinted polymer (NIPs), when difference is to prepare liposome, replace hydrating fluid by the PBS solution that 1ml contains 1mg 6-FAM, all the other steps are identical with the preparation method of SPA-MIPs with NIPs.Then FAM-SPA-MIPs, FAM-NIPs are diluted 5 times with physiological saline, staphylococcus aureus bacterium liquid (1 × 10 respectively with 6-FAM (the 6-FAM concentration of three is identical)
8cFU/mL) 100 times are diluted; Again by FAM-SPA-MIPs, FAM-NIPs or 6-FAM respectively with staphylococcus aureus bacterium liquid by volume for 1:1 mixes, be placed in 37 DEG C of constant temperature water tanks, as a control group with the bacterium of same concentrations simultaneously, to hatch after 6h centrifugal 10min under 7000r/min condition, removing supernatant, retains precipitation, and the fresh culture 5mL adding sterilizing in precipitation mixes, survey fluorescence intensity level with flow cytometer, result as shown in Figure 5.Result shows, after staphylococcus aureus hatches 6h with FAM-SPA-MIPs, FAM-NIPs and 6-FAM respectively, the fluorescence intensity that picked-up bag carries fluorescein preparation is different, bacterium is significantly higher than FAM-NIPs and 6-FAM in conjunction with the amount of FAM-SPA-MIPs, and fluorescence intensity is 2.2 times and 2.8 times of FAM-NIPs and 6-FAM respectively.Show that FAM-SPA-MIPs has stronger external binding ability to staphylococcus aureus.
Secondly, erythromycin medicine carrying being carried out to the molecular engram targeted system constructed by the present invention, by investigating it, its targeting being verified further to the inhibitory action of staphylococcus aureus.Bag carries the imprinted polymer (EM-SPA-MIPs) of erythromycin and the target liposomes (SPA-MIPs) of the preparation method of non-imprinted polymer (EM-NIPs) with reference to SPA trace and the preparation method of non-imprinted polymer (NIPs), difference is when preparing liposome, be that 1:15 takes erythromycin 1mg and is dissolved in 200 μ l ethanol by the mass ratio of erythromycin and liposome, take soybean lecithin 10.5mg, cholesterol 4.5mg is dissolved in chloroform: in the mixed solution 800 μ l of methyl alcohol (1:1), three is all added in eggplant-shape bottle, remaining steps is consistent with the target liposomes (SPA-MIPs) of SPA trace and the preparation method of non-imprinted polymer (NIPs).The concentration determination of erythromycin adopts high performance liquid chromatography (HPLC), and testing conditions is as follows: chromatographic column: C18 post; Column temperature: 25 DEG C; Determined wavelength: 210nm; Mobile phase: 0.1mol/L ammonium dihydrogen phosphate (ADP) buffer solution (pH 6.5 adjusted by triethylamine): acetonitrile volume ratio is 70:30; Flow velocity: 1.0mL/min; Sample size 50 μ L.Testing result shows, and is 100 ~ 500 μ gmL at erythromycin concentration
-1good linear relationship is had between erythromycin concentration (X) and HPLC chromatographic peak area (Y) in scope.Calibration curve equation is: Y=30054X+56254 (R
2=0.9991), basic, normal, high content assaying precision RSD is respectively 3.46%, 2.18% and 1.52%, and the rate of recovery is respectively 96.54 ± 2.05%, 98.73 ± 2.59%, 100.26 ± 3.02%.
Finally, measure erythromycin liposome encapsulation: get erythromycin liposome and be divided into two parts, a through Sephadex G-50 gel column purified pool, another part is directly diluted to same volume, both all add methyl alcohol breakdown of emulsion, measure erythromycin concentration C and C in two increment product respectively
0.Computational envelope rate (EE): EE=C/C
0× 100%.After testing, the envelop rate average (n=3) of erythromycin liposome EM-LIP is 91.25 ± 1.48%, and the envelop rate average (n=3) of erythromycin imprinted polymer (EM-SPA-MIPs) is 86.25 ± 2.53%.
When measuring the fungistatic effect of EM-SPA-MIPs to staphylococcus aureus, according to the incubation method adopted in staphylococcus aureus picked-up experiment, first EM-SPA-MIPs, EM-NIPs, EM (the EM concentration of three is identical) are diluted 5 times respectively with physiological saline, staphylococcus aureus bacterium liquid (1 × 10
8cFU/mL) 100 times are diluted; Again by EM-SPA-MIPs, EM-NIPs, EM respectively with staphylococcus aureus bacterium liquid by volume for 1:1 mixes, be placed in 37 DEG C of constant temperature water tanks, the bacterium of same concentrations as a control group, to hatch after 6h centrifugal 10min under 7000r/min condition, removing supernatant, retain precipitation, the fresh culture 5mL adding sterilizing in precipitation mixes.By sample suspension in 96 orifice plate point samples, be placed in constant incubator and cultivate, and survey its OD value respectively at 0h, 1h, 2h, 4h, 6h, 8h, experimental result as shown in Figure 6.Result shows, the OD value of EM-SPA-MIPs group and EM-NIPs group is all lower than the OD value of non-administration group (Control group), compared with NIL group, the OD of EM-SPA-MIPs group is starkly lower than EM-NIPs group, illustrate that the inhibitory action of EM-SPA-MIPs to bacterium is better than EM-NIPs, the Targeting Effect further illustrating the molecular engram targeting preparation constructed by the present invention is obvious.
The preparation of the target liposomes of embodiment 2, matrix metalloproteinase MMP-2 trace and evaluation thereof
The preparation of the target liposomes of matrix metalloproteinase MMP-2 trace, concrete steps are as follows:
(1) film dispersion ultrasonic method is adopted to prepare liposome, concrete grammar is: take soybean lecithin 10.5mg, cholesterol 4.5mg, dissolve with the solution 1ml that chloroform and methyl alcohol volume ratio are 1:1, then the rotary evaporation that reduces pressure under 37 DEG C of conditions removing organic solvent, form the uniform transparent lipid membrane of one deck, add pure water 1mL in 37 DEG C of aquation 1h, utilize the ultrasonic 5min (800W of ultrasonic cell disrupte instrument, 50 times, interval 10s after each ultrasonic 10s), obtain liposome (LIP);
(2) adopt sol method to prepare the liposome of finishing silica, concrete grammar is: by phosphatide: ethyl orthosilicate (TEOS): H
2o mol ratio be 1:8:344 slowly add in liposome through volume fraction be 10% ethanol dispersion TEOS, then pH to 8.50 is regulated with the sodium hydroxide solution of 0.1mol/L under agitation, room temperature (18-25 DEG C) stirs spends the night, the silica precursor do not deposited in removing system of again mixture being dialysed, obtain the liposome of finishing silica, called after silica liposome (S-LIP);
(3) ethylene linkage modifies silica liposome, concrete grammar is: be slowly added drop-wise to by 3-sulfydryl-1-propane sulfonic acid sodium (MPS) of 50 μ L after disperseing half an hour in the ethanol of 250 μ L, then get its 10 μ L dispersant liquid drop to be added in the liposome of 2ml silica room temperature (18-25 DEG C) and to stir and spend the night, obtain the silica liposome (MS-LIP) that ethylene linkage is modified;
(4) preparation of matrix metalloproteinase MMP-2 imprinted polymer, concrete grammar is: getting acrylamide (AAM) 4mg and N-N methylene-bisacrylamide (MAA) 2mg, to be dissolved into pH be 7.4, concentration is in the PBS of 0.02M, ultrasonic dissolution, obtain mixed solution A, then 2mg matrix metalloproteinase MMP-2 is dispersed in 1mL ethylene linkage modify silica liposome solutions in and be positioned over stirring at room temperature 0.5h, again mixture is carried out vacuum outgas 10min, obtain mixed solution B, getting 200 μ L mixed solution A is under nitrogen protection added dropwise in mixed solution B, and nitrogen blowing 10min, room temperature (18-25 DEG C) stirs lower prepolymerization 1h, 7 μ L ammonium persulfate (APS are dripped under nitrogen protection, 10%, W/W) and 4.5 μ L tetramethylethylenediamine (TEMED, 5%, W/V) initiated polymerization in mixture, and then be positioned over stirring at room temperature 24h, with pure water dialysis one week, removing template molecule and unreacted reagent, freeze-drying is to constant weight and matrix metalloproteinase MMP-2 imprinted polymer called after MMP-2-MIPs.With pure water dialysis one week, removing template molecule and unreacted reagent.Namely freeze-drying to constant weight obtains the target liposomes (MMP-2-MIPs) of matrix metalloproteinase MMP-2 trace.
The synthesis of non-imprinted polymer (NIPs) is not except adding template protein molecule, and other preparation methods are the same.
The adsorption experiment of the target liposomes of matrix metalloproteinase MMP-2 trace: for investigating the target liposomes of the matrix metalloproteinase MMP-2 trace prepared to the specific adsorption ability of matrix metalloproteinase MMP-2, adopt following methods to verify it.
First, FITC fluorescence labeling has been carried out to matrix metalloproteinase MMP-2: get 4mg matrix metalloproteinase MMP-2 and be dissolved in 0.2M Na
2pO
4in solution, separately get 0.16mgFITC and be dissolved in 0.2M Na
2pO
4in solution, the mol ratio of FITC and matrix metalloproteinase MMP-2 is made to be 1.2:1.Under magnetic agitation, the solution containing matrix metalloproteinase MMP-2 is added in the solution containing FITC, lucifuge reaction 1.5h.After reaction terminates, centrifugal 10min under 8000r/min condition, to remove sediment matrix metalloproteinases MMP-2.Get supernatant Sephadex G-50 gel column and carry out separation and purification, freeze-drying, obtain the matrix metalloproteinase MMP-2 of FITC mark.
Dynamic Adsorption is tested: by 5mg MMP-2-MIPs and NIPs respectively with the 0.2mg/ml FITC-MMP-2 solution of 5ml in 37 DEG C of oscillation incubations, 1ml is taken out respectively at 0.25h, 0.5h, 1h, 2h, 4h, with the centrifugal 10min of 4500r/min, measure the fluorescence intensity of supernatant, result as shown in Figure 7.Result shows, and the absorption of MMP-2-MIPs and NIPs to FITC-MMP-2 increases along with the increase of time, namely reaches dynamic equilibrium when 2h.Further, during Dynamic Adsorption balance, MMP-2-MIPs is about 5 times of NIPs to the adsorbance of FITC-MMP-2, illustrates that the molecular engram target polymer carrier that the present invention builds has good adsorption capacity to albumen.
Adsorption isotherm experiment: accurately take 1mg MMP-2-MIPs and NIPs and be placed in centrifuge tube respectively, respectively with FITC-MMP-2 solution oscillation incubation under 37 DEG C of conditions of 0.05mg/ml, 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.8mg/ml of 1ml, take out after 2h, with the centrifugal 10min of 4500r/min, Aspirate supernatant measures fluorescence intensity, and result is as shown in Figure 8.Adsorption isotherm experiment shows, polymer changes along with the change of FITC-MMP-2 concentration the absorption of template molecule, when the concentration of FITC-MMP-2 is lower than 0.2mg/ml, ratio of adsorption rises fast, when the concentration of FITC-MMP-2 reaches 0.2mg/ml, ratio of adsorption reaches balance, and compared with NIPs, MMP-2-MIPs has higher adsorption capacity and selective preferably to FITC-MMP-2.
Specific adsorption is tested: specifically have chosen staphylococcus aureus surface Protein S PA, lysozyme (LYZ), bovine serum albumin(BSA) (BSA), Streptavidin (SA) is competition albumen, the preparation method of the FITC mark reference FITC-MMP-2 of competition albumen, obtain FITC-SPA, FITC-LYZ, FITC-BSA respectively, FITC-SA.The concrete operations of specific adsorption test are: take a series of 1mg MMP-2-MIPs and NIPs and be placed in centrifuge tube respectively, FITC-SPA, FITC-LYZ, FITC-BSA, FITC-SA and FITC-MMP-2 solution adding the 0.2mg/ml of 1ml to be placed on oscillator after vibration absorption 2h in 37 DEG C, measure the fluorescence intensity in supernatant respectively, result as shown in Figure 9.Result shows, and MMP-2-MIPs only has higher absorption property to MMP-2, and relatively low to the absorption property of other competition albumen, similar with the absorption property of NIPs to MMP-2.This illustrates that the imprinted polymer MMP-2-MIPs constructed by the present invention has higher specific selectivity to template molecule MMP-2.
On this basis, the target liposomes of matrix metalloproteinase MMP-2 trace is carried out to the inspection on cellular level, due to the one that MMP-2 type matrix metalloproteinase enzyme is tumour cell outer matrix metalloproteinases enzyme, therefore, its trace ability is evaluated by the targeting of checking MMP-2 imprinted polymer to tumour cell.In this experiment, select tumour cell U937, fluorescence probe is adriamycin.
The target liposomes (DOX-MMP-2-MIPs) carrying the matrix metalloproteinase MMP-2 trace of adriamycin and bag carry target liposomes (MMP-2-MIPs) and the non-imprinted polymer (NIPs) of preparation method with matrix metalloproteinase MMP-2 trace of the non-imprinted polymer (DOX-NIPs) of adriamycin, when difference is to do liposome, ammonium sulphate gradient is adopted to prepare the liposome of bag adriamycin: to take soybean lecithin 10.5mg, cholesterol 4.5mg, dissolve with the solution 1ml that chloroform and methyl alcohol volume ratio are 1:1, then the rotary evaporation that reduces pressure under 37 DEG C of conditions removing organic solvent, form the uniform transparent lipid membrane of one deck, add pure water 1mL in 37 DEG C of aquation 1h, utilize the ultrasonic 5min (800W of ultrasonic cell disrupte instrument, 50 times, interval 10s after each ultrasonic 10s), obtain liposome (LIP), gained LIP to be dialysed 4h to water.With the Doxorubicin solution of 0.9% normal saline 10mg/ml, then the Doxorubicin solution of the LIP after dialysis and 150 μ l is hatched 20min 60 DEG C of conditions, through the adriamycin that the removing of Sephadex G-50 gel column is free after having hatched, the liposome (DOX-LIP) carrying adriamycin must be wrapped.
Then measure U937 cell to test the picked-up of DOX-MMP-2-MIPs and DOX-NIPs, evaluate the targeting of the target liposomes of the trace constructed by the present invention.Concrete operations are: by 10^
4u937 cell chulture spend the night, 2h is hatched respectively with DOX-SPA-MIPs, DOX-NIPs (the DOX concentration of the two is identical), then wash off not by the liquid of cellular uptake with PBS, then 30min is fixed with paraformaldehyde, cell after fixing dyes through DAPI, the picked-up result of observation of cell under fluorescence microscope, result as shown in Figure 10.Result shows, can be observed stronger red fluorescence around the nucleus of hatching with DOX-MMP-2-MIPs, and the red fluorescence arrived with the cell observation that DOX-NIPs is hatched is more weak, thus, can absolutely prove, DOX-MMP-2-MIPs has better external binding ability to U937 cell.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (10)
1. based on the preparation method of the target liposomes of protein molecular trace, it is characterized in that, comprise the steps: to get acrylamides and N-N methylene-bisacrylamide is in the PBS solution or water that 1 ~ 5:1 is dissolved to pH6.8 ~ 7.4, concentration is 0.02 ~ 0.1M in mass ratio, ultrasonic, obtain mixed liquor A, in gained mixed liquor A, the total concentration of acrylamides and N-N methylene-bisacrylamide is 1 ~ 5mg/ml; Olefinic recycle key modifies silica liposome, and be then scattered in by protein molecular in the silica liposome solutions of ethylene linkage modification and stir, vacuum outgas obtains mixed liquid B; Under nitrogen protection, mixed liquor A is added dropwise in mixed liquid B for 1:1 ~ 1:10 by volume, nitrogen blowing, stirs prepolymerization; then ammonium persulfate and tetramethylethylenediamine initiated polymerization is dripped under nitrogen protection; continue to stir, dialysis, must based on the target liposomes of protein molecular trace.
2. preparation method according to claim 1, it is characterized in that, the preparation method of described silica liposome is: first adopt film dispersion ultrasonic method to prepare liposome, then in liposome, add the ethyl orthosilicate through ethanolic solution dispersion, pH to 8 ~ 10.4 are regulated with sodium hydroxide solution under stirring, stirring is spent the night, and dialysis, obtains silica liposome.
3. preparation method according to claim 2, is characterized in that: the addition of described ethyl orthosilicate is by phosphatide: ethyl orthosilicate: H
2the mol ratio of O is 1:8:344.
4. preparation method according to claim 2, is characterized in that: the volume fraction of described ethanolic solution is 5% ~ 25%.
5. preparation method according to claim 2, it is characterized in that: the method that described film dispersion ultrasonic method prepares liposome is that phosphatide and cholesterol to be dissolved in volume ratio be in the chloroform of 1:1 and the mixed solution of methyl alcohol, decompression rotary evaporation removing organic solvent, form transparent lipid membrane, add water hydratable, ultrasonic, obtain liposome.
6. preparation method according to claim 5, is characterized in that: the mol ratio of described phosphatide and cholesterol is 2 ~ 3:1.
7. preparation method according to claim 5, is characterized in that: described ultrasonic condition is under 800W condition ultrasonic for 50 times, and each ultrasonic 10s, continues ultrasonic after the 10s of interval.
8. preparation method according to claim 5, is characterized in that: the condition of described aquation is 37 DEG C of Water Underizations 1 hour.
9. the preparation method according to any one of claim 1 ~ 8, is characterized in that: described protein molecular is staphylococcus aureus protein A or matrix metalloprotease MMPs.
10. the target liposomes based on protein molecular trace obtained by the preparation method described in any one of claim 1 ~ 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410755690.1A CN104492396B (en) | 2014-12-10 | 2014-12-10 | Preparation method of target liposomes based on protein molecular trace and products thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410755690.1A CN104492396B (en) | 2014-12-10 | 2014-12-10 | Preparation method of target liposomes based on protein molecular trace and products thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104492396A true CN104492396A (en) | 2015-04-08 |
| CN104492396B CN104492396B (en) | 2016-08-17 |
Family
ID=52933898
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410755690.1A Expired - Fee Related CN104492396B (en) | 2014-12-10 | 2014-12-10 | Preparation method of target liposomes based on protein molecular trace and products thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104492396B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106349283A (en) * | 2016-08-23 | 2017-01-25 | 陕西源邦生物技术有限公司 | Preparation method of high-purity phosphatidylserine |
| CN110540589A (en) * | 2019-01-08 | 2019-12-06 | 西南大学 | Polypeptide, polypeptide-modified lipid carrier and application |
| CN114748644A (en) * | 2022-05-20 | 2022-07-15 | 齐齐哈尔大学 | Preparation method of phycocyanin molecularly imprinted drug carrier with ZIF-8 as matrix |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005063835A1 (en) * | 2003-12-31 | 2005-07-14 | Council Of Scientific & Industrial Research | Cobalt imprinted polymer composition, preparation and use thereof |
| CN101669909A (en) * | 2008-09-12 | 2010-03-17 | 复旦大学 | Biological adhesive liposome preparation for eyes and preparation method thereof |
| CN104146987A (en) * | 2014-07-28 | 2014-11-19 | 陕西师范大学 | Simple preparation method of lipidosome/silicon dioxide composite nanocapsules |
-
2014
- 2014-12-10 CN CN201410755690.1A patent/CN104492396B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005063835A1 (en) * | 2003-12-31 | 2005-07-14 | Council Of Scientific & Industrial Research | Cobalt imprinted polymer composition, preparation and use thereof |
| CN101669909A (en) * | 2008-09-12 | 2010-03-17 | 复旦大学 | Biological adhesive liposome preparation for eyes and preparation method thereof |
| CN104146987A (en) * | 2014-07-28 | 2014-11-19 | 陕西师范大学 | Simple preparation method of lipidosome/silicon dioxide composite nanocapsules |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106349283A (en) * | 2016-08-23 | 2017-01-25 | 陕西源邦生物技术有限公司 | Preparation method of high-purity phosphatidylserine |
| CN110540589A (en) * | 2019-01-08 | 2019-12-06 | 西南大学 | Polypeptide, polypeptide-modified lipid carrier and application |
| CN110540589B (en) * | 2019-01-08 | 2021-07-20 | 西南大学 | Polypeptide, polypeptide-modified lipid carrier and application |
| CN114748644A (en) * | 2022-05-20 | 2022-07-15 | 齐齐哈尔大学 | Preparation method of phycocyanin molecularly imprinted drug carrier with ZIF-8 as matrix |
| CN114748644B (en) * | 2022-05-20 | 2023-08-18 | 齐齐哈尔大学 | Preparation method of phycocyanin molecularly imprinted drug carrier with ZIF-8 as matrix |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104492396B (en) | 2016-08-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ansari et al. | Molecularly imprinted polymers for capturing and sensing proteins: Current progress and future implications | |
| Karaseva et al. | Synthesis and application of molecularly imprinted polymers for trypsin piezoelectric sensors | |
| Yang et al. | One-pot synthesis of hydrophilic molecularly imprinted nanoparticles | |
| Ma et al. | Luminescent molecularly-imprinted polymer nanocomposites for sensitive detection | |
| Chianella et al. | Direct Replacement of Antibodies with Molecularly Imprinted Polymer Nanoparticles in ELISA Development of a Novel Assay for Vancomycin | |
| Yang et al. | Direct and highly selective drug optosensing in real, undiluted biological samples with quantum-dot-labeled hydrophilic molecularly imprinted polymer microparticles | |
| Thongchai et al. | A microflow chemiluminescence system for determination of chloramphenicol in honey with preconcentration using a molecularly imprinted polymer | |
| Gianneli et al. | Label-free in-flow detection of receptor recognition motifs on the biomolecular corona of nanoparticles | |
| Horak et al. | Albumin‐coated monodisperse magnetic poly (glycidyl methacrylate) microspheres with immobilized antibodies: application to the capture of epithelial cancer cells | |
| JP2011516819A (en) | Detection apparatus and method | |
| Kang et al. | Mussel-inspired universal bioconjugation of polydiacetylene liposome for droplet-array biosensors | |
| Liu et al. | Flow-through immunosensors using antibody-immobilized polymer monoliths | |
| Ma et al. | Oriented surface epitope imprinted polymer-based quartz crystal microbalance sensor for cytochrome c | |
| CN104492396A (en) | Targeted liposome based on protein molecular imprinting and preparation method thereof | |
| Qiao et al. | Monolith and coating enzymatic microreactors of L-asparaginase: kinetics study by MCE–LIF for potential application in acute lymphoblastic leukemia (ALL) treatment | |
| Sut et al. | Supported lipid bilayer coatings: Fabrication, bioconjugation, and diagnostic applications | |
| Kovaleva et al. | Acid–base properties of nanoconfined volumes of anodic aluminum oxide pores by EPR of pH-sensitive spin probes | |
| KR20140092029A (en) | Hydrogel bead based biosensor | |
| Jung et al. | Porosity-Tuned Chitosan–Polyacrylamide Hydrogel Microspheres for Improved Protein Conjugation | |
| He et al. | Recognition and analysis of biomarkers in tumor microenvironments based on promising molecular imprinting strategies with high selectivity | |
| US10502735B2 (en) | System and apparatus for porously-encapsulated magnetic-nanoparticle biosensors | |
| KR100934646B1 (en) | Bioanalytical Microfluidic System Based on Aspheric Hydrogel Microparticles | |
| Heider et al. | Structural characterization of individual vesicles using fluorescence microscopy | |
| JP5099787B2 (en) | Quantitative analysis method | |
| CN110441184A (en) | A kind of preparation method of the polymer-modified QCM-D sensor of solid phase trace |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160817 Termination date: 20161210 |